Sterility in male mouse F1 hybrids obtained by crossing different species is genetically determined. Previous studies have implicated four hybrid sterility (Hst) loci on proximal Chr 17, as well as pairing problems in the pseudoautosomal region (PAR) that prevents normal segregation of Chrs X and Y. We have been studying loci associated with hybrid sterility on proximal Chr X. One locus, Ihtw1, was discovered using a congenic hybrid strain C57BL/6J.SPRET-Hprta(AT24), containing 17 map units from M. spretus Chr X on a C57BL/6 background. Ihtw1 is associated with low testis weight and allows partial fertility, but only half of the testicular tubules are filled with developing sperm, while the other tubules are empty. We have developed subcongenic strains from AT24 and defined a critical interval of 1 map unit containing Ihtw1. Using a cross between two subcongenic strains we have obtained a strain with small testes, carrying only the critical interval from M. spretus. Another study involves loci, also on proximal Chr X, found in our analysis of progeny from the cross (C57BL/6J x M. macedonicus) x C57BL/6J. Half of the male progeny from this cross did not progress to meiotic metaphase I, a more severe phenotype than found with M. spretus. This study also identified a locus on proximal Chr 17, whose relationship to the previously described Hst loci needs to be determined. Genetic isolation of the phenotypes is a necessary first step toward identification and cloning of the genes. Congenic strains have been made in which proximal Chrs X and 17 from M. macedonicus have been separately placed in a C57BL/6 background. This proposal has two major goals. In Aim 1 we propose to refine the genetic map around Ihtw1, generate a BAC contig and use the sequence from the orthologous region of human Chr X to identify candidate genes. The current best candidates are Fgf13, Hspa9b, Pdcd8 and Sox3. A series of tests are designed to identify the gene encoded by Ihtw1. A screen for suppressor loci will be continued using the cross (AT24 x M. spretus) x C57BL/6 and performing a genome wide screen on progeny males whose testis weights will be used for QTL analysis. In Aim 2 we will focus mainly on the M. macedonicus meiotic metaphase I project, with less emphasis on Chr 17, and propose to make subcongenic strains from the congenic strains to further localize the genes involved. A search for autosomal suppressors of the phenotype will use an approach similar to that used for Ihtw1. The loci on proximal Chr X and their suppressors may play significant roles in causing sterility in the human male. They are also probably involved in early stages of speciation.